Lipoproteins play critical role in human health (1-4). Studies in mice have shown that alterations in high-density lipoprotein (HDL) receptor SR-BI expression lead to defects in biliary cholesterol secretion, female fertility, red blood cell development, as well as contributing to the development of atherosclerosis and coronary heart disease (1). Significantly, macrophage type-I and type-II class-A scavenger receptors (SR-A), responsible for uptake of modified low-density lipoprotein (mLDL), have been implicated in the pathological deposition of cholesterol during atherogenesis (2). Thus, the uptake of mLDLs by macrophages with subsequent release of immune mediators and formation of lipid-laden foam cells can lead to the development of atherosclerosis (3). Uptake of native and modified lipoproteins by macrophages and other cell types is an attractive therapeutic target (4-5). However, high-throughput approaches allowing identification of agents that can modulate lipoprotein uptake by macrophages and other cells are lacking.
Current methods rely on labeling of lipoproteins with non-pH-sensing traditional dyes such as BODIPY FL™, DiI and Alexa Fluor™. Assays using such dyes are characterized by low signal-to-noise ratios. Additionally, traditional dyes are susceptible to fluorescence quenching observed in the aggressive and acidic environment of the uptake vesicles (6). Furthermore, these assays are not easily amenable to high-throughput schemes and assays of non-adherent cell types due to requirement for multiple washes needed to achieve accurate measurement of lipoprotein uptake.
Provided herein are methods for lipoprotein labeling with pH-sensing dyes (e.g., CypHer5E™) that demonstrate superior signal-to-noise ratio compared to traditional dyes. Also provided are high-throughput assays for measuring lipoprotein uptake and screening methods for identifying agents that affect lipid accumulation in cell types carrying lipoprotein receptors.